Coxiella targets mononuclear phagocytes during in vivo infection. Thus, infection of cultured primary or immortalized human monocytes/macrophages is considered the most physiologically relevant in vitro model of Coxiella-host cell interactions. Because of the presence of various endolysosomal markers, the Coxiella parasitophorous vacuole (PV) is considered phagolysosome-like. However, the degradative properties of the mature PV are unknown and there are conflicting reports on the maturation state and growth permissiveness of PV harboring virulent phase I or avirulent phase II Coxiella in human mononuclear phagocytes. To address these questions, we employed infection of primary human monocyte-derived macrophages (HMDM) and THP-1 cells as host cells to directly compare PV maturation kinetics and pathogen growth in cells infected with the Nine Mile phase I (NMI) or phase II (NMII) variants of Coxiella. In both cell types, phase variants replicated with similar kinetics, achieving roughly 2-3 logs of growth before reaching stationary phase. HMDM infected by either phase variant secreted similar amounts of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-&#945;. In infected THP-1 cells, an equal percentage of NMI and NMII PV decorated with the early endosomal marker Rab5, the late endosomal/lysosomal markers Rab7 and CD63, and the lysosomal marker cathepsin D at early (8 h) and late (72 h) time points post-infection (PI). Mature PV (2-4 days PI) harboring NMI or NMII contained proteolytically-active cathepsins and quickly degraded E. coli. These data suggest that Coxiella does not actively inhibit phagolysosome function as a survival mechanism. Instead, NMI and NMII resist degradation to replicate in indistinguishable digestive PV that fully mature through the endolysosomal pathway. These data also indicate infection of human macrophages with biosafety level 2 phase II Coxiella is an accurate model to investigate host-pathogen interactions. We have previously demonstrated that the PV membrane is cholesterol-rich and that inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. However, the precise source(s) of PV membrane cholesterol is unknown as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. Coxiella lacks enzymes for de novo cholesterol biosynthesis;however, the organism encodes a eukaryotic-like &#8710;24 sterol reductase homolog, CBU1206. Rare in prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under control of a galactose-inducible promoter. Erg4 is a yeast delta24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-GFP, a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide, and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during Coxiella intracellular growth. Because cholesterol metabolism inhibitors can have pleiotropic effects, we utilized cholesterol-free DHCR24-/- mouse embryo fibroblasts (MEFs) to determine if Coxiella indeed requires cholesterol for intracellular growth. These cells lack the &#8710;24 sterol reductase required for the final reduction step in cholesterol biosynthesis and consequently accumulate desmosterol in cellular membranes. Desmosterol cannot replace cholesterol in lipid rafts, membrane microdomains that play important regulatory roles in endocytosis, cellular signaling, and intracellular trafficking. Coxiella infection efficiency, PV phenotype and growth rate in DHCR24-/- and wildtype MEFs were similar, suggesting that 1) desmosterol can substitute as the primary membrane sterol, or 2) Coxiella can generate cholesterol from host cell precursors. In control experiments, the intracellular pathogen Chlamydia trachomatis also grew in the absence of cholesterol, though possibly with a delayed developmental cycle. Collectively, these data indicate that cholesterol and lipid rafts are not essential for Coxiella and Chlamydia infection. Ongoing experiments are investigating whether the PV in cholesterol-free cells has altered biological properties. We have idnetified 34 Coxiella Dot/Icm type IV secretion system (T4SS) substrates that represent a treasure trove of potential virulence factors. Elucidation of their cellular activities and targets will provide needed information on the Coxiella/host relationship. Because all Coxiella Dot/Icm substrates were initially identified using Legionella as surrogate, we developed reporter systems using the shuttle vector pJB-Cat/Kan (Project AI000946) to directly confirm secretion by Coxiella. We currently have Coxiella transformants expressing T4SS effectors fused to the enzymatic reporter proteins cyaA and blaM, and to epitope tags FLAG and HA. It is hoped that over-expressed epitope-tagged Coxiella effectors will traffic normally and allow detection of their subcellular localization by immunofluorescence. An interesting subset of six effectors is encoded by the Coxiella cryptic QpH1 plasmid. Using new genetic tools, secretion of plasmid effectors by Coxiella during host cell infection was confirmed using &#946;-lactamase and adenylate cyclase translocation assays and a C-terminal secretion signal was indentified. When ectopically expressed in HeLa cells, plasmid effectors traffic to different subcellular sites, including autophagosomes, ubiquitin-rich compartments, and the endoplasmic reticulum. Collectively, these results suggest Coxiella plasmid-encoded T4SS substrates play important roles in subversion of host cell functions, thereby providing a plausible explanation for the absolute maintenance of plasmid genes by this pathogen. Ectopic expression in mammalian cells of chromsomally-encoded effectors fused to fluorescent proteins also reveals a variety of subcellular localizations including microtubules and the Coxiella PV membrane. Yeast two-hybrid analysis has revealed potential eucaryotic binding partners for six Coxiella Dot/Icm substrates. These preliminary results now set the stage for defining effector function. A biphasic developmental cycle whereby highly resistant small cell variants (SCV) are generated from large cell variants (LCV) is considered fundamental to Coxiella virulence. Previous work from our lab revealed that LCV is the replicative form of Coxiella, and that SCV and LCV are compositionally and antigenically different. Further molecular and biochemical analyses of SCV and LCV morphogenesis is necessary to better understand the physiological relevance of Coxiella biphasic development. However, the intracellular growth of Coxiella imposes considerable experimental constraints. Therefore, we investigated development in our new host cell-free growth medium, Acidified Cysteine Citrate Medium (ACCM). Biphasic development in ACCM was indistinguishable from Coxiella propagated in vivo. The fidelity of Coxiella morphogenesis in ACCM now provides ample pure cell populations for biochemical studies, ultrastructural analyses, and phenotyping.